Mechanism Of DNA Methylation Modifies MdMYB1 Promoter To Regulate Anthocyanin Transport In Apple

Fruit color is one of the important agronomic characters and important attributes of fruit quality.Anthocyanin is the main pigment that determines the fruit color,and it is beneficial to human’s health.Therefore,it is the pursuit for breeders to cultivate apple varieties with good fruit coloration and high anthocyanin content.As a way of breeding,sports selection is an excellent way to improve the quality and agronomic characters of apple varieties,such as coloring.MYB1/10 promoter is methylated by DNA methylation affecting the expression of MYB1/10 to regulate the transcript level of the downstream genes of anthocyanin pathway,finally,the difference of sports color occurred.However,it is unclear that how DNA methylation recognizes the MYBl/10 site to affect the expression ofMYB1/10 and regulate other downstream genes of anthocyanin pathway.Therefore,it will provide theoretical support for apple sports breeding to use apple color sports mutant to study how DNA methylation affects apple MdMYBl promoter to explore the mechanism of apple sports formation.In this study,we selected three ’Fuji’ apple varieties as the material,they were ’Nagafu 2’,’Yanfu 3’ and ’ Yanfu 8’.Among them,’Yanfu 3’ is the red sport of ’Nagafu 2’ and ’Yanfu 8’ is the red sport of ’Yanfu 3’.The anthocyanin content of three ’Fuji’ apple,the methylation level of MdMYBl promoter,the genes regulating MdMYBl promoter methylation were identified,and the downstream gene of anthocyanin pathway of MdMYBl were identified.The main results are listed as follows1.The anthocyanin gradually accumulated from 4 to 16 days after bag removed,and reached the peak at 16 days forming red stripe phenotype in ’Nagafu 2’.Anthocyanin accumulated rapidly in ’Yanfu 3’ and ’Yanfu 8’,’Yanfu 3’ and finally inhibited the deep red stripe,while’Yanfu 8’ showed full red phenotype.The anthocyanin content in sports was higher than their parents.The expression of anthocyanin main regulatory gene,MdMYBl,was analyzed.The results showed that the transcription level of MdMYBl in the lighter colored varieties(such as’Nagafu 2’)was slightly increased,while the transcription level of MdMYBl in the deeply colored varieties(such as ’Yanfu 3’ and ’Yanfu 8’)increased significantly at the later stage of development.The differential expression of MdMYBl gene was the main reason for the different color of the three ’Fuji’ apple varieties2.The cDNA and gDNA sequences of MdMYBl in three ’Fuji’ apple varieties were analyzed The results showed that there was no base mutation and mismatch between cDNA and gDNA sequences,which indicated that the difference of coloration among the three ’Fuji’ apple varieties was not caused by the genetic variation of MdMYBl.Through detecting the methylation level of MdMYBl promoter of three ’Fuji’ varieties,we found that the MR3 region of MdMYB1 promoter showed significant difference among the three varieties.Further analysis showed that the difference of CHH methylation level in MR3 region of the three ’Fuji’ varieties was the main reason for the different coloration of the three ’Fuji’ varieties3.Y1H assay was used to verify if the MdAOG4s protein bind to MdMYBl promoter.Y1H results showed that MdAOG4s could bind to MdMYBl promoter.Further Y1H assay showed that MdAG04s could only bind to the p4 region of MdMYBl promoter.The promoter interaction between MdAOG4s and MdMYBl was also verified by ChIP-qPCR assay.The results showed that the c3 fragment could be enriched in the calli of 35S::AG04s-GFP,and it was proved that MdAG04s could bind to the c3 fragment of MdMYBl promoter in vivo.The interaction between MdAG04s,MdDRM2s and MdRDMl was verified by Co-IP assay.The results show that MdAG04-1 can interact with MdDRM2-1/2 and MdRDMl,MdAG04-2 can interact with MdDRM2-1/2 and MdRDMl,and MdDRM2-1/2 can interact with MdRDMl Meanwhile,the interaction between MdAG04s,MdDRM2s and MdRDMl was also proved by Pull-down assay in vitro.The results showed that MdAG04s,MdDRM2s and MdRDMl could interact with each other and form protein complex.Overexpression of MdAG04s and MdDRM2s in ’Orin’ and red-flesh calli could increase the methylation level of MdMYB1/10 promoter.4.We used ChIP-qPCR assay to further verify whether MdAG04s can bind to methylated MR3 regions,and found that MdAG04s can bind to methylated MR3 region.Subsequently,RNAi interfered the expression of MdNRPEl,the largest subunit in PolV,ChIP-qPCR assay showed that the binding of MdAG04s to MR3 region was suppressed after inhibition of MdNRPEl.The results show that the MdAG04s bound to CHH region requires the lncRNAs5.In this study,EMSA assay was used to verify whether MdAG04 binds to MdMYBl promoter in vitro.EMSA assay showed that MdAG04s only binds to the probe2 sequence of c3 fragment,and does not bind to probel,probe3,probe4.The results of piecewise mutation showed that MdAG04s could bind to 2-ml,2-m2,2-m4 and 2-m5,but not to 2-m3.The results showed that the complete m3 sequence(GATATCAGAC)was necessary for MdAG04s binding to MdMYBl promoter.Further EMSA assays were carried out with MdFLS probe(containing ATATCAGA sequence)and MdGST probe(containing GATATCA sequence)probes.The results showed that MdAG04s did not bind to MdGST probes,but bound toMdFLS probes,even it was two bases less than GATATCAGAC.In summary,MdAG04s can specifically bind ATATCAGA sequences.ChIP-PCR results show that the binding of MdAG04s to c3 region does not affect after silencing the MdNRPEl.In conclusion,MdAG04s can directly bind to MdMYBl promoter through ATATCAGA sequence6.69 GST coding genes were searched from the apple genome.MdGSTF6 was identified as a candidate gene involved in anthocyanin pathway.The analysis of homologous phylogenetic tree showed that apple MdGSTF6 was a homologous gene of PpRiant in peach,FvRAP in strawberry,VvGSTF12 in grape,AtGSTF12 in Arabidopsis thaliana.Amino acid sequence analysis showed that the sequence ofMdGSTF6 was the most similar to that of PpRiant,FvRAP,VvGSTFl2.Among them,since peach PpRiant encodes an anthocyanin transporter,MdGSTF6 may be a candidate protein for apple anthocyanin transporter7.Through subcellular localization and membrane cytosolic protein isolation localization,MdGSTF6 was localized on the vacuolar membrane of the cell.Transgenic complementary Arabidopsis tt09 could be able to rescue its phenotype of anthocyanin accumulation.In apple peel,using virus-induced gene silencing assay,inhibiting the expression of MdGSTF6 could affect anthocyanin accumulation.Using RNAi,silencing MdGSTF6 expression in tissue-cultured ’Gala’ seedlings affected anthocyanin accumulation.Silencing MdGSTF6 did not influence the expression of anthocyanin regulatory and structural genes.In summary,MdGSTF6 is an anthocyanin transporter and plays an important role in anthocyanin accumulation.8.YIH screening was performed from the apple fruit cDNA library using the MdGSTF6 promoter as bait.Through sequencing and blast,results show that MdMYB1 can act on the upstream of MdGSTF6.This result was verified by YIH assay.EMSA assay showed that MdMYB1 could bind to the MBS motif of MdGSTF6 promoter.ChIP-PCR assay showed that MdMYB1 interacted with the promoter of MdMYBl in vivo.LUC reporter assay showed that expression of MdGSTF6 could be activated by and regulated by MBW complex.GUS staining further showed that MdMYB 1 could activate the promoter activity of MdGSTF6 and upregulate the expression of MdGSTF6.